医学免疫学实验：4 B淋巴细胞膜表面免疫球蛋白的检测—直接荧光法

1、BackgroundMaterials and MethodsResultsDiscussion2SmIg 是B细胞的抗原识别受体，也是B细胞特异的表面标志，用直接免疫荧光法可检查出SmIg。用荧光素标记的抗Ig 抗体，在一定条件下与淋巴细胞混合，荧光素标记的抗Ig 抗体可以与B 细胞表面的Ig 结合，在荧光显微镜下观察可见B 细胞膜上出现荧光。此方法可用来鉴定B 淋巴细胞。3实验材料实验材料:ICR 小鼠FITC标记兔抗小鼠Ig抗体Hanks 液 (pH 7.4, 0.1% NaN3)离心管，移液器离心机4实验方法实验方法准备细胞6取脾于6ml Hanks液中研磨轻柔混匀，吸取1ml细胞到离

2、心管1000rpm 离心，5min6Methods准备细胞免疫荧光标记荧光显微镜观察加入 FITC标记抗体 100l4 避光，孵育30min 洗2遍取脾于6ml Hanks液中研磨轻柔混匀，吸取1ml细胞到离心管1000rpm 离心，5min光镜荧光显微镜B cells percentage in splenic lymphocyte = N(Fluo)N(Light)118256= 44.5%7B cells percentage in splenic lymphocyte44.5%Reported value:40%Fell A H, Preston P M. Int. J. Parasi

3、tol., 1992, 22(4): 491-501.55% (BALB/c mouse)Pellegrini A, Guiaz N, Aoki M P, et al. Int. immunol., 2007, 19(12): 1395-1402.8Experimental mistakes analysisPipetting too many times in washing step Weak signalExposure to light Weak signalSmear too thick in specimen preparation View cannot matchNo cont

4、rol group False positive移液多次洗涤步骤微弱信号；曝光微弱信号；涂抹太厚不易观察；没有对照组假阳性Direct ImmunofluorescenceIndirect ImmunofluorescenceAdvantage Shorter sample staining time Simpler dual and triple labeling procedureAdvantage Greater sensitivity Commercially produced secondary antibodies are Inexpensive, availableDisadva

5、ntage Lower signal Less flexibility Higher costDisadvantage Potential for cross-reactivity9Application of Immunofluorescence12Application of immunofluorescenceDirect immunofluorescence (DIF)Skin/mucosaBullous pemphigoidPemphigus vulgarisLupusRenalPulmonary transplantationIndirect immunofluorescence

6、(IIF)Pemphigus vulgarisParaneoplastic pemphigusBullous and/or cicatricialpemphigoidOther fields of applicationsBone marrowCell surface markersInfective disordersViral (Influenza virus, EBV, adeno virus, herpes virus, varicella zoster, CMV and HIV)Bacterial (M. Pneumoniae, M. Tuberculosis, T. Pallidi

7、um)Parasitic (Entamoeba, Giardia, Cyclospora)Amyloid, Mucin, LipidArvind Babu R S, Chandrasekar P, Chandra K L, et al. J. Orofac. Sci., 2013, 5(1): 2.Limitations of immunofluorescenceFactors affect the quality of a fluorescence image:Nonspecific antibody binding / dilute antibodyNot proper handling of the biopsy specimenSignal is weakened due to exposure to light.10Dermatologic disease diagnosis:Immunofluore